Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum)

A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed...

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Bibliographic Details
Published in:Physiologia plantarum 2008-06, Vol.133 (2), p.242-253
Main Authors: Van Riet, Liesbet, Altenbach, Denise, Vergauwen, Rudy, Clerens, Stefan, Kawakami, Akira, Yoshida, Midori, Van den Ende, Wim, Wiemken, Andres, Van Laere, André
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Biological and medical sciences
Chromatography, Ion Exchange
Cloning, Molecular
DNA, Complementary - genetics
Enzymes
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Plant
Glycoside Hydrolases - chemistry
Glycoside Hydrolases - genetics
Glycoside Hydrolases - isolation & purification
Glycoside Hydrolases - metabolism
Hydrolysis
Metabolism
Molecular Sequence Data
Phylogeny
Pichia - enzymology
Plant physiology and development
Sequence Alignment
Sequence Homology, Amino Acid
Substrate Specificity
Sucrose - pharmacology
Triticum - enzymology
Triticum - genetics
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Summary:A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed mainly β2-1 linkages in fructans and was inhibited by sucrose. A cDNA could be obtained after reverse transcriptase polymerase chain reaction (RT-PCR)-based strategies and screening of a cDNA library. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed that the encoded protein has essentially the same characteristics as the native enzyme. Homology with previously described 1-FEH isoforms from wheat was high (97% identity), and the enzyme showed minor differences to the previously published enzymes. The relative abundance of 1-FEH transcripts in different tissues was investigated by using quantitative RT-PCR.
ISSN:0031-9317
1399-3054
DOI:10.1111/j.1399-3054.2008.01070.x